The sympathetic nervous system plays a central role in cardiovascular regulation in both health and disease.^1-5 The sympathetic nervous system can increase peripheral vascular resistance and cardiac output to raise blood pressure. Arteriolar vasoconstriction, as well as sympathetic-mediated venoconstriction (with consequent central redistribution of blood and increased cardiac output), both act to increase blood pressure. Cardiac sympathetic chronotropic and inotropic effects also increase blood pressure, particularly in the setting of increased vascular resistance. Thus, sympathetic traffic to the peripheral vasculature and sympathetic discharge to the heart have complementary effects on blood pressure. Activation of the sympathetic nervous system may also contribute to blood pressure levels in the long term by other mechanisms, by its effects on the kidney, on the renin-angiotensin system, on blood vessel growth and permeability, and via resetting of the arterial baroreflex. Sympathetic activation has been implicated in the pathogenesis of hypertension, coronary artery disease, cardiac arrhythmias, and heart failure. This review focuses on methods for studying sympathetic activity in humans.

Measurements of urine and plasma noradrenaline

Traditionally, activity of the sympathetic nervous system was assessed using measurements of urine noradrenaline and adrenaline or their precursors and metabolites. However, this “static” approach cannot provide reliable assessment of short-term changes in sympathetic activity and, therefore, has been replaced by measurement of plasma noradrenaline concentration. These measurements provide useful information, but also have significant limitations.⁶ Firstly, circulating noradrenaline represents only a small fraction (5% to 10%) of the amount of neurotransmitter secreted from nerve terminals. Secondly, plasma levels of noradrenaline are influenced, in addition to the level of sympathetic neural outflow, by prejunctional modulation of neurotransmitter release, and the clearance, metabolism, and uptake of noradrenaline from the circulation. Thus, plasma measurements do not allow discrimination between central (increased secretion) or peripheral (reduced clearance) mechanisms of increased levels of the neurotransm tter.⁵ Thirdly, the use of plasma noradrenaline is based on the assumption that these measurements reflect “overall” sympathetic activity. Contrary to this assumption, there are profound regional differences in the activity and control of sympathetic function. Furthermore, the reproducibility and sensitivity of plasma noradrenaline values are lower than those of microneurographic recordings.⁷ The value of plasma catecholamine measurements is enhanced if they are combined with assessment of responses to adrenergic antagonists and agonists. Using this approach, it has been shown that mildly hypertensive individuals had elevated plasma noradrenaline levels, augmented decreases in vascular resistance in response to α-adrenergic blockade, and no increase in α-receptor sensitivity as assessed by responses to noradrenaline.⁸ This study demonstrated augmented sympathetic vasoconstrictor activity in young mildly hypertensive humans, suggesting that increased sympathetic vasoconstriction results from enhanced sympathetic neural release of noradrenaline, and not from augmented α-adrenergic response to the neurotransmitter.

Noradrenaline spillover rate measurements

The radiotracer noradrenaline kinetic technique (noradrenaline “spillover”) avoids the confounding influence of neurotransmitter clearance and permits assessment of noradrenaline spillover from specific target organs.⁹ Hypertension, in particular “early” hypertension, may be characterized by increased sympathetic traffic not only to the heart and blood vessels, but also to the kidneys. Using measurements of noradrenaline spillover, Esler et al¹⁰ found that noradrenaline was elevated in hypertensive patients, particularly in young hypertensives, and that the increased spillover occurred mainly from the heart and kidneys.
Using jugular vein noradrenaline spillover measurements, Ferrier et al¹¹ reported that higher sympathetic activity in hypertension may be explained by increased cerebral noradrenaline release. This increased noradrenaline release was confined to subcortical forebrain regions. The same group of investigators subsequently reported that subcortical noradrenaline release was linked with total body noradrenaline spillover as well as renal noradrenaline spillover.¹² Since the forebrain is involved in the emotional responses (especially the defense reaction) it has been suggested that increased noradrenaline spillover from certain subcortical regions may represent a neurochemical manifestation of stress.
Quantitative assessment of tritiated noradrenaline uptake from plasma has demonstration of impairment of noradrenaline transporter function in essential hypertension.¹³ The potential role of impaired neuronal noradrenaline reuptake can be directly assessed by infusion of the noradrenaline transport inhibitor desipramine.¹⁴ Finally, noradrenaline stores in the human heart could be estimated by quantifying the processing inside sympathetic nerves of tritiated noradrenaline to its intraneuronal metabolite, tritiated dihydroxyphenylglycol (DHPG), coupled with measurement of the specific activity of DHPG in coronary sinus plasma.^15,16

Microneurography

Direct intraneural recordings using microneurography provide a moment-to-moment measure of central sympathetic neural outflow independent of the influence of the neuroeffector junction. This technique involves the recording of multiunit sympathetic nerve discharge from a peripheral nerve, usually the peroneal nerve.^17,18 Sympathetic nerve activity is recorded using tungsten microelectrodes (shaft diameter 200 µm, tapering to an uninsulated tip of 1 to 5 µm) inserted selectively into muscle or skin fascicles. Recently, microneurographic approach also allowed quantification of single-fiber muscle sympathetic nerve traffic.^19,20
Microneurography permits separate recordings of sympathetic nerve activity to muscle circulation (MSNA) or skin (SSNA). MSNA reflects the vasoconstrictor signal to the skeletal muscle vasculature, is acutely sensitive to blood pressure changes, and is closely regulated by the arterial and cardiopulmonary baroreflexes. SSNA is not altered by either arterial or cardiopulmonary baroreflexes. At rest, in a room-temperature environment, SSNA reflects vasomotor neural traffic to skin blood vessels with little if any sudomotor activity present.²¹ MSNA and SSNA differ markedly with regard to morphology (Figure 1). SSNA bursts are broad-based and may extend over several cardiac cycles. The duration of each MSNA burst is limited by the cardiac cycle.
Measurement of sympathetic nerve activity from peripheral nerves in humans has been shown to be safe, accurate, quantifiable, and reproducible.²² Also important is the fact that simultaneous measurements of sympathetic nerve activity from different limbs show identical profiles in terms of burst frequency and morphology. Thus, recordings in one limb can be reliably assumed to reflect recordings of sympathetic nerve activity to the muscle vascular bed throughout the body.²³
The neural signals were amplified, filtered, rectified, and integrated to obtain a voltage display of sympathetic nerve activity. Sympathetic bursts are identified by a careful visual inspection of the voltage neurogram or by dedicated software. Muscle sympathetic nerve activity can be expressed as bursts per minute and bursts per 100 heartbeats, which allows comparison of sympathetic discharge between individuals (Figure 2). The amplitude of each burst can also be determined, and sympathetic activity may be calculated as bursts/minute multiplied by mean burst amplitude and expressed as units/minute. Measurement of nerve activity at baseline before each intervention are expressed as 100%. Changes in integrated MSNA allow evaluation of within-subject changes in sympathetic traffic in response to different stressors during the same recording session. Figure 3 illustrates changes in MSNA in response to activation and deactivation of arterial baroreceptors was achieved by intravenous infusion of nitroprusside and phenylephrine. Baroreflex control of MSNA is estimated by calculating percent changes in the integrated activity associated with changes in MAP during infusion of the drugs.
The advent of microneurography has enabled a direct evaluation of the reflex sympathetic neural response to chemoreflex stimulation. These studies have documented that the peripheral and central chemoreflexes have powerful effects on sympathetic activity in both health and disease, and may contribute importantly to disease pathophysiology, particularly in conditions such as hypertension,²⁴ obstructive sleep apnea,²⁵ and heart failure.²⁶

Figure 1. Recordings of skin and muscle sympathetic nerve activity in a normal subject. While the duration of each muscle (baroreflex-dependent) sympathetic nerve activity burst is limited by the cardiac cycle, skin (baroreflex-independent) sympathetic nerve activity bursts are broad based and may extend over several cardiac cycles. (Adapted from ref 5 with the permission of the publisher).	Figure 2. Recordings of muscle sympathetic nerve activity ilustrating low (top) and high (bottom) activity. (Adapted from ref 5 with the permission of the publisher).
Figure 3. Spectral analysis of simultaneous recordings of RR variability in a control subject (left) and in a patient with hypertension (right). There is a relative predominance of the LF component over the HF component of RR interval in the patient with hypertension. (Adapted from ref 5 with the permission of the publisher).	Figure 4. Changes in muscle sympathetic nerve activity during intravenous infusion of nitroprusside (top) and phenylephrine (bottom) in a control subject. (Adapted from ref 5 with the permission of the publisher).

Power spectral analysis

Spectral analysis of heart rate variability is a widely used noninvasive technique for assessment of autonomic indices of neural cardiac control.²⁷ In normal humans, short-term RR interval variability occurs predominantly at a low frequency (0.04 to 0.14 Hz) and a high frequency (±0.25 Hz, synchronous with the respiratory frequency) (Figure 4). The respiratoryrelated HF component is attributed mainly to vagal mechanisms. By contrast, different hypotheses have been proposed for the LF oscillation of RR variability. In several studies, the LF component was not related to rates of noradrenaline spillover from the heart and or muscle sympathetic nerve traffic.⁶ Thus, while the LF/HF ratio may be considered as a marker of sympathovagal balance, it is unjustified to consider the low frequency power as a surrogate measure of sympathetic nerve firing.

Imaging techniques

Several imaging methods have recently been introduced to assess sympathetic activity in humans. These techniques utilizing both positron emission tomography and single photon-emission computed tomography scanning were used to evaluate the anatomy of sympathetic innervation. The most widely used scanning agents include [¹²³I]metα-iodobenzylguanidine (MIBG), 6-[¹⁸F]fluorodopamine, and [¹¹C]hydroxyephedrine.⁶ This methods demonstrated sympathetic denervation in patients with pure autonomic failure.

Summary

Development of new methods briefly reviewed in this article was instrumental for better understanding of the sympathetic neural mechanisms of cardiovasculardisease, and ultimately lead to remarkable improvements in clinical management. However, it has been stressed that even state-of-art methods such as microneurography or noradrenaline spillover cannot viewed as the “gold standard.” The limitations and disadvantages of the various techniques have been reviewed in greater detail elsewhere.⁶ Several methods should be viewed as being complementary. The limitations of these techniques can be significantly reduced if they are used in combination.
Application of the discussed methods is limited to laboratory settings, and it is rather unlikely that they will be used in clinical practice. However, there is growing evidence that tachycardia is a reliable marker of high sympathetic tone, and cardiovascular risk (Figure 5). Faster heart rate predicts the development of sustained hypertension in subjects with borderline elevated blood pressure values.²⁸ Tachycardia is associated with increased risk of cardiovascular morbidity and mortality.²⁸ Therefore, despite intrinsic limitations, assessment of heart rate should be recommended as an additional method of stratifying cardiovascular risk, which may help in choosing optimal therapy.

Figure 5.
Mechanisms and consequences of tachycardia. (Adapted from ref 5 with the permission of the publisher).

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